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Infection and Immunity, December 2006, p. 7021-7023, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.00977-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Monocyte Chemoattractant Protein 1 Does Not Contribute to Protective Immunity against Pneumococcal Pneumonia

Mark C. Dessing,^1,2* Alex F. de Vos,^1,2 Sandrine Florquin,³ and Tom van der Poll^1,2

Center for Infection and Immunity Amsterdam (CINIMA),¹ Center for Experimental and Molecular Medicine,² Department of Pathology, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands³

Received 19 June 2006/ Returned for modification 31 July 2006/ Accepted 7 September 2006

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To determine the role of monocyte chemoattractant protein 1 (MCP-1) during pneumococcal pneumonia, MCP-1 knockout and wild-type mice were infected with Streptococcus pneumoniae. Pulmonary MCP-1 levels were strongly correlated to bacterial loads in wild-type mice. However, MCP-1 knockout and wild-type mice were indistinguishable with respect to bacterial growth, inflammatory responses, and lethality.

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Streptococcus pneumoniae is the most frequently isolated causative pathogen in community-acquired pneumonia (2, 4). Previous studies examined the role of several cytokines in host defense against pneumococcal pneumonia (9, 12, 15-17), but knowledge of the role of chemokines is limited. Monocyte chemoattractant protein 1 (MCP-1) is a chemokine produced during infection and inflammation (3, 7, 11) which primarily attracts monocytes and memory T cells (5) but, during severe bacterial infection, may also contribute to neutrophil recruitment (10, 13). In addition, MCP-1 has been found to exert anti-inflammatory effects during murine endotoxemia (18). In a model of acute nonlethal pneumonia caused by Pseudomonas aeruginosa, treatment with anti-MCP-1 resulted in increased neutrophil influx into the lungs and enhanced lung injury without influencing the clearance of Pseudomonas (1). In a lethal pneumococcal pneumonia model, anti-MCP-1 treatment did not influence the accumulation of either neutrophils or macrophages in the lungs; the impact on the growth of pneumococci or lethality was not reported (6).

To further investigate the role of MCP-1 in pneumococcal pneumonia, we infected 10- to 11-week-old MCP-1 knockout (KO) C57BL/6 mice (Jackson Laboratory, Bar Harbor, Maine) and sex- and age-matched C57BL/6 wild-type (WT) mice (Charles Rivers, Maastricht, The Netherlands) with various doses of S. pneumoniae serotype 3 (ATCC 6303; American Type Culture Collection, Rockville, MD). All experiments were approved by the Animal Care and Use Committee of the University of Amsterdam (Amsterdam, The Netherlands). Mice were inoculated intranasally with 50 µl containing 4 x 10³ to 50 x 10³ CFU S. pneumoniae as described earlier (9, 12). Blood and lungs were obtained and processed for immunoassays and quantitative cultures as described previously (9, 12). MCP-1, tumor necrosis factor alpha (TNF-), and interleukin-6 (IL-6) were measured by cytometric bead array multiplex assay (BD Biosciences, San Jose, CA). Macrophage inflammatory protein 2 (MIP-2) and cytokine-induced neutrophil chemoattractant (KC) were measured by enzyme-linked immunosorbent assay (R & D Systems, Abingdon, United Kingdom). Myeloperoxidase was measured by enzyme-linked immunosorbent assay (HyCult, Uden, The Netherlands). Hematoxylin- and eosin-stained lung slides were analyzed for bronchitis, edema, interstitial inflammation, pleuritis, endothelialitis, and intra-alveolar inflammation. Each parameter was graded on a scale from 0 to 4, with 0 as absent and 4 as severe. The total "lung inflammation score" was expressed as the sum of the scores for each parameter. MLE-12 mouse alveolar epithelial cells (10⁵/ml in RPMI 1640 supplemented with 5 mg/liter insulin, 10 mg/liter transferrin, 5 µg/liter sodium selenite, 10 nM hydrocortisone, 10 nM ß-estradiol, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2% fetal bovine serum; Sigma) and primary mouse peritoneal macrophages (10⁵/ml in RPMI 1640 supplemented with 1 mM pyruvate, 2 mM L-glutamine, penicillin, streptomycin, and 10% fetal bovine serum) were incubated overnight with 1 x 10⁷ CFU heat-killed S. pneumoniae (HKSP, 30 min at 70°C) or medium alone, and MCP-1 was measured in the supernatant. Statistics were analyzed by using Mann-Whitney U test. The difference in positive blood culture between groups was analyzed by chi-square test. For survival analyses, Kaplan-Meier analysis followed by log rank test was performed. Correlations between pulmonary bacterial load and MCP-1 concentrations were calculated by Spearman's rank correlation test. Values are expressed as means ± standard errors of the means (SEM). A P value of 0.05 was considered statistically significant.

At 48 h after infection of WT mice with various doses (4 x 10³ to 50 x 10³ CFU) of S. pneumoniae, lung MCP-1 levels were strongly correlated with the pulmonary bacterial load (Fig. 1A) (P < 0.0001, R² = 0.8228). Mice with positive blood cultures had significantly higher levels than nonbacteremic mice (972 ± 312 versus 56 ± 15 pg/ml, respectively; P < 0.0001). To further investigate which cell types produce MCP-1 during pneumococcal pneumonia we stimulated MLE-12 alveolar epithelial cells and primary macrophages with HKSP. Stimulation with HKSP significantly increased MCP-1 production in both cell types (Fig. 1B). To evaluate the role of MCP-1 in host defense against pneumococcal pneumonia, we determined the bacterial load in lung homogenates prepared 5, 24, and 48 h after infection with 5 x 10⁴ S. pneumoniae CFU. MCP-1 KO and WT mice displayed similar bacterial outgrowth and occurrence of bacteremia (Fig. 2A). Also during less overwhelming infection (10⁴ or 4 x 10³ CFU, 48 h), no significant differences in bacterial outgrowth in the lungs of MCP-1 KO and WT mice were observed (Fig. 2B and C). After infection with 10⁴ S. pneumoniae CFU, more MCP-1 KO than WT mice had a positive blood culture at 48 h, suggesting that MCP-1 may reduce the systemic spread of pneumococci (P = 0.05); however, such an effect was not seen after infection with 5 x 10⁴ or 4 x 10³ CFU. To determine whether this difference was of biological relevance, we repeated these experiments but found no significant difference in mortality (Fig. 2D and E). Lung inflammation scores, determined 48 h after infection with 5 x 10⁴ or 10⁴ S. pneumoniae CFU, were similar in WT and MCP-1 KO mice (5 x 10⁴ CFU, 12.4 ± 2.6 versus 9.4 ± 1.3, respectively, P = 0.57; 10⁴ CFU, 3.7 ± 1.5 versus 4.8 ± 0.5, respectively, P = 0.10). In addition, histopathologic analysis and pulmonary myeloperoxidase levels revealed similar granulocyte influx in WT and MCP-1 KO mice (data not shown). Cytokines and chemokines play an important role in an adequate antibacterial defense in bacterial infections (8, 14). Thus, we determined the levels of the cytokines TNF- and IL-6 and the chemokines MIP-2 and KC in whole-lung homogenates and cytokine concentrations in plasma obtained from WT and MCP-1 KO mice after infection with 5 x 10⁴ S. pneumoniae CFU (Fig. 3). Although several pulmonary cytokine and chemokine concentrations tended to be lower in MCP-1 KO mice, especially at 48 h after infection, differences were not significantly different (P > 0.25). Similarly, no differences between WT and MCP-1 KO mice were detected after infection with the two lower bacterial doses (data not shown).

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FIG. 1. MCP-1 production. (A) Correlation between pulmonary MCP-1 levels and bacterial load during pneumococcal pneumonia. MCP-1 levels and bacterial loads in whole lung homogenates from WT mice 48 h after inoculation with 4 x 103 to 50 x 103 S. pneumoniae CFU. The closed line represents curve fit; the dashed line represents the 95% confidence band. Goodness of fit is presented as R2. (B) MCP-1 production in MLE-12 cell line and mouse macrophages. MLE-12 and mouse macrophages (M) incubated with either medium (black bars) or HKSP (white bars). *, P < 0.05. Data are means ± SEM (n = 4 to 5 per group).

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FIG. 2. MCP-1 deficiency does not influence bacterial growth or survival during pneumococcal pneumonia. (A) Bacterial loads in whole-lung homogenates 5, 24, and 48 h after inoculation with 5 x 104 S. pneumoniae CFU in WT (black bars) and MCP-1 KO mice (open bars). Bacterial loads in whole-lung homogenates (B and C) and survival (D and E) of WT (black bars or symbols) and MCP-1 KO mice (open bars or symbols) inoculated with 104 CFU (B and D) or 4 x 103 CFU (C and E). BC+ indicates the number of positive blood cultures. Data are means ± SEM (n = 6 to 8 per group [A, B, and C]; n = 10 to 12 per group [D and E]).

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FIG. 3. MCP-1 deficiency does not influence cytokine or chemokine concentrations. Levels of cytokines (IL-6, TNF-) and chemokines (KC, MIP-2) in whole-lung homogenates and cytokine concentrations in plasma obtained from WT (black bars) and MCP-1 KO (white bars) mice after infection with 5 x 104 S. pneumoniae CFU. Data are means ± SEM (n = 6 to 8 per group).

In conclusion, we demonstrate that pulmonary MCP-1 production is correlated to the bacterial growth during pneumonia caused by S. pneumoniae. MCP-1 deficiency did not influence the host response after infection with several doses of S. pneumoniae, suggesting that endogenous MCP-1 does not play a major role in the pathogenesis of pneumococcal pneumonia. Of note, this conclusion only applies for the specific (serotype 3) bacterial strain and the model used here. An earlier study showed that administration of an anti-MCP-1 antibody did not have impact on leukocyte recruitment to the lungs after infection with S. pneumoniae, whereas the combined treatment with antibodies directed against MCP-1, MIP-1, and RANTES reduced the influx of macrophages/monocytes (6). Together with our current results, these data suggest that during pneumococcal pneumonia the lack of MCP-1 may be compensated for by other mediators.

 
We thank Joost Daalhuisen and Marieke ten Brink for technical assistance during the animal experiments and Regina de Beer for preparations of lung tissue slides. MLE-12 cells were kindly provided by Jeffrey Whitsett, Division of Pulmonary Biology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati.

We declare that we have no competing interests.

M.C.D. conducted all in vivo experiments and wrote the manuscript. S.F. analyzed all histology slides and took part in writing the manuscript. A.F.D.V. performed cell stimulation experiments and took part in writing the manuscript. T.V.D.P. designed and supervised the study and took part in writing the manuscript.

This study was funded by an institutional grant to M.C.D.

 
* Corresponding author. Mailing address: Center for Experimental and Molecular Medicine, Room F0-117, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. Phone: 31205665247. Fax: 31206977192. E-mail: m.c.dessing{at}amc.uva.nl.

Published ahead of print on 18 September 2006.

Editor: J. N. Weiser

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Infection and Immunity, December 2006, p. 7021-7023, Vol. 74, No. 12
0019-9567/06/$08.00+0     doi:10.1128/IAI.00977-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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